Molecular Biology of Pneumocystis Carinii and its Clinical Application

卡氏肺孢子虫是一种机会致病菌，常引起因艾滋病、癌症化疗或器官移植免疫抑制治疗而免疫抑制或免疫缺陷的患者发生致命性肺炎。卡氏肺孢子虫是一种真核微生物，感染许多哺乳动物宿主。卡氏肺孢子虫的连续体外培养尚未建立，因此大量培养的困难阻碍了该生物体研究的进展。尽管如此，有必要对卡氏肺孢子虫进行分子研究。表面糖蛋白。卡氏肺孢子虫的主要表面免疫决定簇是一组称为P115的蛋白质。它们由六种主要的等电变体组成，并被甘露糖高度糖基化。抗P115单克隆抗体的频繁产生暗示了这些分子的强抗原活性。去糖基化分析表明，P115的糖基部分在抗原活性中起着关键作用. PI 15中 ...更多信息 cDNA克隆。用pMDagtII载体构建卡氏肺孢子虫cDNA文库。用抗P115蛋白部分的抗体筛选文库，并分离出40个阳性克隆。核苷酸序列分析显示，它们被归类为至少三个相似但不相同的组。它们与单克隆抗体共享相同的表位位点。此外，针对这些cDNA-lacZ融合蛋白之一的兔多克隆抗体与完整的P115分子反应。因此，我们确信我们克隆了P115 cDNA。观察到的cDNA结构的异质性可能反映了卡氏肺孢子虫生物体的异质性或其他寄生原生动物表面结构的变异性。聚合酶链反应诊断。我们已经成功地利用聚合酶链反应（PCR）方法检测卡氏肺孢子虫微生物通过扩增5S rDNA序列在各种临床和动物样本。该检测方法灵敏度高、特异性强。广泛的试验证明了PCR方法在肺炎患者痰液样本中的有效性。PCR阳性的患者，然后每天与喷他脒或磺胺甲恶唑/甲氧苄啶，并通过PCR检测进行监测。这些试验证明了应用PCR监测卡氏肺孢子虫肺炎治疗的有效性。_减

Pneumocystis carinii is an opportunistic pathogen which often causes fatal pneumonia in patients under immunosuppressed or immune deficient conditions due to AIDS, cancer chemotherapy or immunosuppressive therapy for organ transplantation. P. carinii is a eukaryotic microorganism and infects many mammalian hosts. Continuous in vitro culture of P. carinii has not been established and thereby the difficulty of mass cultivation impedes progress in the study of this organism. Nonetheless, it is necessary to pursue molecular approaches to P. carinii studies.1. Surface Glycoproteins. The major surface immunodeterminants of P. carinii is a group of proteins called P115. They are composed of six major isoelectric variants and highly glycosylated with mannose. The frequent generation of anti-Pll5 monoclonal antibodies implicates a strong antigenic activity of these molecules. Deglycosylation analysis revealed that the sugar moiety of P115 plays a crucial role in the antigenic activity.2. PI 15 … More cDNA Cloning. P. carinii cDNA library was constructed using lambdagtll vector. The library was screened with antibodies against the P115 protein moiety, and 40 positive clones were isolated. Nucleotide sequence analyses revealed that they were classified to at least three similar but not identical groups. They share the same epitope site to monoclonal antibodies. Furthermore, rabbit polyclonal antibodies raised against one of these cDNA-lacZ fusion proteins reacted with the intact P115 molecules. Therefore, we were convinced of our cloning of P115 cDNA. The observed heterogeneity in the cDNA structure might reflect heterogeneity in P. carinii organisms or variability of the surface structure as found in other parasitic protozoa.3. Diagnosis with Polymerase Chain Reaction. We have successfully utilized the polymerase chain reaction (PCR) method to detect P. carinii organisms by amplifying the 5S RDNA sequence in various clinical and animal samples. The detection was highly sensitive and specific. Extensive trials manifested efficacy of the PCR method in sputum samples from patients who suffered from pneumonia. The PCR-positive patients were then administered daily with pentamidine or sulfamethoxazole/trimethoprim and were monitored by the PCR assay. These trials demonstrated the efficacy of applying PCR to the monitoring of therapy to P. carinii pneumonia. _ Less